Relaxin-like factor and methods and uses thereof

ABSTRACT

The present invention relates to a relaxin-like factor, its derivatives or analogs, and uses thereof. The present invention further relates to compositions comprising a relaxin-like factor, its derivatives or analogs, and relaxin wherein such composition exhibits an additive or synergistic effect.

This application is a continuation of application Ser. No. 09/041,491,filed Mar. 12, 1998, now abandoned, which application is a divisional ofSer. No. 08/484,219, filed Jun. 7, 1995, now U.S. Pat. No. 5,911,997,both of which are incorporated herein by reference in their entirety andto which application we claim priority under 35 U.S.C. §120.

A portion of the work set forth herein was supported by grantsNIHGMS-48893 and NSF MCB-9406656 and by the Medical University of SouthCarolina.

1. INTRODUCTION

The present invention relates to a relaxin-like factor, its derivativesor analogs, and uses thereof. The present invention further relates tocompositions and formulations comprising a relaxin-like factor, itsderivatives or analogs, and relaxin wherein such composition exhibits anadditive or synergistic effect.

2. BACKGROUND OF THE INVENTION

A family of hormones, comprising insulin, insulin-like growth factors (Iand II), bombyxin, molluscan insulin-related peptide and relaxin, hasbeen identified and designated as “insulin-related.” Blundell andHumbel, 1980, Nature 287:781-787; Büllesbach and Schwabe, 1991, J. Biol.Chem. 266:10754-10761. The proteins comprising this family of hormonesrepresents a group of polypeptides having homologous primary andsecondary structure but divergent biological functions.

Relaxin has been purified from a variety of species including porcine,murine, equine, shark, tiger, rat, dogfish and human. In the human,relaxin is most abundantly found in the corpora lutea (CL) of pregnancy.Mature human relaxin is a hormonal peptide of approximately 6000 daltonswhich facilitates the birth process by remodelling the reproductivetract before parturition. More specifically, relaxin appears to mediatethe restructuring of connective tissues in target organs to obtain therequired changes in organ structure during pregnancy and parturition.See, Hisaw, 1926, Proc. Soc. Exp. Biol. Med. 23:661-663; Schwabe, etal., 1977, Biochem. Biophys. Res. Comm. 75:503-570; James, et al., 1977,Nature, 267:544-546. A concise review of relaxin was provided bySherwood, D. in The Physiology of Reproduction, Chapter 16, “Relaxin”,Knobil, E. and Neill, J., et al. (eds.), (Raven Press Ltd., New York),pp. 585-673 (1988).

While predominantly a hormone of pregnancy, relaxin has also beendetected in the non-pregnant female as well as in the male.Bryant-Greenwood, 1982, Endocrine Reviews 3:62-90; Weiss, 1984, Ann.Rev. Physiol. 46:43-52.

Two human gene forms encoding for human relaxin have been identified,(H1) and (H2). Hudson, et al., 1983, Nature 301 628-631; Hudson, et al.,1984, EMBO J., 3:2333-2339; and U.S. Pat. Nos. 4,758,516 and 4,871,670.Only one of the gene forms (H2) has been found to be transcribed in CL.It remains unclear whether the (H1) form is expressed at another tissuesite, or whether it represents a pseudo-gene. When synthetic humanrelaxin (H2) and certain human relaxin analogs were tested forbiological activity, the tests revealed a relaxin core necessary forbiological activity as well as certain amino acid substitutions formethionine that did not affect biological activity. Johnston, et al., inPeptides: Structure and Function, Proc. Ninth American PeptideSymposium, Deber, C. M., et al. (eds.) (Pierce Chem. Co. 1985).

Methods of making relaxin are described in U.S. Pat. No. 4,835,251 andU.S. Pat. No. 5,464,756 (PCT US90/02085) and PCT US94/06997. Methods ofusing relaxin in cardiovascular therapy and in the treatment ofneurodegenerative diseases are described in U.S. Pat. No. 5,166,191 andin PCT US92/06927. Certain formulations of human relaxin are describedin U.S. Pat. No. 5,451,572.

The structure and biological function and activity of the remainingmembers of the insulin-related family have been extensively studied.See, e.g. Robinson and Fritz, 1981, Biol. Reprod. 24:1032-1041; Soder,et al., 1992, Endocrinology 131:2344-2350; Luthman, et al., 1989, Eur.J. Biochem 180(2):259-65; Jhoti, et al., 1987, FEBS Lett. 219:419-425;Smit, et al., 1988, Nature 331:535-538. Among the structural featuresshared between relaxin and the remaining members of the insulin-relatedfamily of hormones are molecular weight, a “two-chain” structurecomprising a B-chain, a connecting C-peptide, and an A-chain, and thenumber and disposition of disulfide links.

Despite these similarities, the proteins comprising the insulin-relatedfamily have been found to have distinct biological functions andactivities. It has been reported that this distinction is in large parta consequence of differences between a few type-specific amino acidresidues. For example, the difference between the glycine in positionA14 of human type II relaxin and the isoleucine in the equivalentposition (A10) of insulin is considered critical in distinguishingbetween the biological-activity of the two proteins. Schwabe andBüllesbach, 1994, FASEB J. 8:1-2.

A protein having the structural characteristics of insulin, insulin-likegrowth factor (IGF) and relaxin has been isolated recently from Leydigcells of the testes. Burkhardt, et al., 1993, Genomics 20:13-19. Thisprotein, designated as a Leydig cell-specific insulin-like peptide (LeyI-L), has been characterized as being “insulin-like” due to the genomiclocation of the gene encoding Ley I-L vis a vis the gene encodinginsulin (as compared to the genomic location of the gene encoding eitherrelaxin or IGF). Burkhardt, et al., 1993, Genomics 20:13-19.

The Ley I-L protein has been characterized also as insulin-like, ratherthan either IGF-like or relaxin-like, based upon the protein's C-peptidechain length. More specifically, the C-peptide length of the Ley I-Lprotein is 49 amino acids, as compared to the 35 amino acid length ofproinsulin C-peptide, the twelve amino acid length of the, known proIGFC-peptides and the over one-hundred amino acid C-peptide length ofprorelaxin. Finally, Ley I-L has been designated insulin-like based onthe observation that the protein is expressed exclusively in prenataland postnatal testicular Leydig cells. Burkhardt, et al., supra.

On the basis of the protein's similarities to insulin and the source ofsuch protein, it was reported that the Ley I-L protein is implicated intesticular function. Id., Adham, et al., 1993, J. Bio. Chem.268(35):26668-6672.

In consultation with the inventors of the present invention, Tashima, etal., 1995, J. Clin. Endocrinal. Metab. 80:707-710, have investigated theaccuracy of previous reports providing that the Ley I-L gene was onlyexpressed in Leydig cells. Specifically, Tashima, et al. investigatedwhether the Ley I-L gene was present and expressed in femalereproductive tissues, the human corpus luteum, trophoblasts, fetalmembranes and breast tissue. As with the case with H2 relaxin, Tashima,et al. determined that the Ley I-L protein can be found in human corpusluteum and trophoblast. Unlike H2 relaxin, however, Ley I-L was notfound to be expressed in fetal membranes, decidua and breast tissue.

Neither the Burkhardt/Adham group nor the Tashima group have reportedthe biological function of the Ley I-L protein. Thus, while thestructure of this putative Ley I-L protein has been identified, nocorrect activity or use was known for this protein until the presentinvention, which completed the discovery of RLF through theidentification and proof of its utility.

3. SUMMARY OF THE INVENTION

The present invention is directed to synthesized or recombinantcompositions derived from the deduced amino acid and nucleic acidsequences for human Ley I-L. In one embodiment of the present invention,the composition comprises the full-length amino acid sequence forrelaxin-like factor (RLF). In another embodiment of the presentinvention, the composition comprises a RLF protein derivative whereinthe protein is shortened at either or both its 3′ and 5′ ends of eitheror both the A- or B-chains. In one embodiment, the A chain may be asshort as fifteen amino acids in length and the B chain may be as shortas thirteen amino acids in length. In yet further embodiments of thepresent invention, the composition is radiolabelled or represents ananalog of RLF having relaxin-like activity.

The present invention is further directed to the use of such compoundsfor the treatment of diseases and disorders which may be otherwisetreated with relaxin, either alone, or in combination with relaxin orother relaxin-like agents, and formulations thereof. In one embodimentof the present invention, the diseases or disorders are related to theabnormal expression of collagen and/or fibronectin. More specifically,such diseases or disorders include scleroderma and rheumatoid arthritis.In another embodiment of the present invention, the diseases and/ordisorders are more generally related to the activation of one or morebiological functions as a result of binding with the relaxin or RLFreceptor. Such diseases and/or disorders may include cardiovasculardisease, sinus bradycardia, neurodegenerative or neurologic disease,depression and hair loss.

The present invention is also related to the use of RLF, whetherlabelled or unlabelled, as a tracer which could then be used toseparate, by HPLC, the different RLF derivatives to yield a carrier-freetracer, in binding assays, and for RLF receptor mapping.

4. DESCRIPTION OF THE DRAWINGS

FIG. 1. FIG. 1 depicts the primary structure of the relaxin-like factor(SEQ ID NO:3 and SEQ ID NO:4), as compared with the sequences of humanrelaxin (SEQ ID NO:2 and SEQ ID NO:5) and insulin (SEQ ID NO:1 and SEQID NO:6) wherein the relative positions of the B-chain arginines in RLF,as compared to relaxin, is highlighted.

FIG. 2. FIG. 2 depicts a schematic of the site-directed sequentialdisulfide link formation. Specifically, the schematic providesinformation regarding: 1) trifluoroacetic acid (TFA) deprotection; 2)oxidation of the thiols using DMSO/50 mM NH₄HCO₃ (1:2 v/v); 3)HF-deprotection of Cys(4-methylbenzyl); 4) combination of A and B chainpH 4.5 in 8 M guanidinium chloride; 5) formation of the third disulfidelink by reaction with iodine in 70% acetic acid; 6) liberation of thetryptophan side chain with 10% piperidine; 7) reduction of methioninesulfoxide with a 33 fold excess of NH₄I in 90% TFA.

FIG. 3. FIG. 3 depicts the HPLC record of the purified RLF.Chromatography was performed on Synchropak RP-P (4.1×250 mm) using alinear gradient from 20-50% in 30 min (A:0.1% TFA in H₂O and B:0.1% TFAin 80% acetonitrile) at a flow rate of 1 ml/min.

FIG. 4. FIG. 4 depicts a comparison of the CD spectra of human relaxin,human RLF, and porcine relaxin.

FIG. 5. FIG. 5 depicts the elution record of the HPLC separation of anRLF tracer preparation. The largest peak is unmodified RLF and theshaded region is the major radioactive peak used as tracer.Chromatography was performed on Aquapore 300 (2.1 mm×30 mm) using alinear gradient from 23% B to 34% B over 60 min (A: 0.1% TFA in H₂O andB: 0.1% TFA in 80% acetonitrile) at a flow rate of 0.1 ml/min.

FIG. 6. FIG. 6 depicts the tissue distribution of RLF receptors infemale estrogen primed mice as measured in vitro in a receptor-bindingassay.

FIG. 7. FIG. 7 depicts the bioactivity of an increasing amount ofrelaxin in the presence and absence of 5 μg of RLF per animal. Theincrease in symphyseal width was easily recognized.

FIG. 8. FIG. 8 depicts the bioactivity of an increasing amount of RLF inthe presence of a uniform amount of human relaxin again shows relaxinenhancement.

FIG. 9. FIG. 9 depicts a comparative bioassay of relaxin, RLF, and anoptimal dose of both. RLF alone does not cause symphyseal widening butthe high dose of the mixture still improves upon the high dose ofrelaxin alone.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. Definitions

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not.

The term “effective amount” means a dosage sufficient to providetreatment for the disease state being treated. This will vary dependingon the patient, the disease and the treatment being effected.

The term “relaxin” means human relaxin, including full length relaxin ora portion of the relaxin molecule that retains biological activity andother active agents with relaxin-like activity, such as agents thatcompetitively displace bound relaxin from a receptor. Relaxin can bemade by any method known to those skilled in the art, preferably asdescribed in U.S. Pat. No. 4,835,251 and in U.S. Pat. No. 5,464,756 (PCTUS90/02085) and PCT US94/06997.

5.2. Relaxin-Like Factor: Structure and Activity

RLF shares primary and secondary structural homology with relaxin,insulin and the other members of the insulin-related family of hormones.As reported previously, RLF is structurally closer to insulin thanrelaxin. The deduced primary structure of RLF is set forth at FIG. 1(SEQ ID NOS:3 and 4).

Contrary to early reports, however, the biological function and activityof RLF is similar to relaxin and distinct from insulin. For example,notwithstanding the shift in amino acid sequence of the receptorinteracting region between RLF and relaxin, RLF interacts with the mousebrain receptor to which relaxin binds.

A basis for the present invention is the inventors' unexpected discoverythat the previously isolated but uncharacterized RLF protein bindsspecifically to crude membrane preparations of mouse uterus and brainand shows crossreactivity with the relaxin receptor, but not the insulinreceptor.

The deduced amino acid sequence for RLF would have predicted an oppositeresult because the critical two Arg residues separated by three aminoacids sequence in RLF is offset toward the C-terminal end of the B chainby exactly one turn of the helix. Thus, although RLF projects thearginines at nearly right angles away from the molecular surface in themanner of relaxin, one would expect that shifting the wholereceptor-binding site would present quite a different bindingenvironment to the receptor.

Notably, although RLF binds to the relaxin receptor, it does not appearto competitively bind to the relaxin receptor, vis a vis relaxin, exceptat higher concentrations. Rather, RLF appears to stimulate relaxinresponse. Thus, RLF can play an important supportive role for therelaxin action in humans. In addition, preliminary experiments suggestthat RLF plays a role independent of relaxin in the male gonads.

In addition, although relaxin-like activity has historically beenconsidered in terms of softening the pubic and cervical ligaments inpreparation for parturition, it has also been shown to directly effectcells outside of the reproductive system. For example, consistent withrelaxing RLF may also be instrumental in inhibiting collagen and/orfibronectin overexpression and diseases related thereto (e.g.scleroderma)

Moreover, although RLF possesses relaxin-enhancing properties, asdescribed herein, RLF possess independent and potentially additionalbiological activity.

5.3. RLF Derivatives and Analogs

Following the present identification of RLF as a protein havingrelaxin-like (rather than insulin-like) activity, to the extent that RLFshares primary and secondary homology to relaxin, as well as insulin,identification of biologically active derivatives and analogs of relaxinevidences the identity of biologically active RLF derivatives andanalogs. Active relaxin analogs and derivatives have been identified toinclude, for example, shortening either or both the 5′ and 3′ end of theprotein. See e.g., U.S. Pat. No. 5,023,321. The present invention istherefore directed to biologically active RLF derivatives wherein the 5′and/or 3′ end of the protein has been shortened. See, above referencedpatents.

Importantly, it has been observed in human relaxin that the arginines atpositions B13 and B17 and potentially the amino acids of the first helixturn in the midregion of the B-chain (Arg-Glu-Leu-Val-Arg) (amino acidresidues 13 to 17 of SEQ ID NO:5) are necessary or important to relaxinactivity. Other RLF analogs and derivatives may be obtained using knowntechniques and this structural information regarding relaxin.

Whether the RLF derivative or analog possesses relaxin-like activityand/or utility may be determined using assays known in the art fordetecting relaxin activity. For example, bioassays used for themeasurement of active relaxin during pregnancy and non-pregnancy, asdescribed in Steinetz et al., 1960, Endocrinology 67:102-115 and Sarosiet al., 1983, American Journal of Obstetrics and Gynecology 145:402-405,may be used.

Similarly, specific immunoassays to detect for the presence of proteinshaving relaxin-like activity may also be used. See e.g., Sherwood etal., 1975, Endocrinology 107:691-696; O'Bryne and Steinetz, 1976,Proceedings of the Society for Experimental Biology and Medicine152:272-276. The presence and activity of synthetic analogs of humanrelaxin comprising one or more accessible tyrosines (permitting directiodination) may also be tested using a radioimmunoassay (RIA). Eddie etal., 1986, Lancet 1:1344-1346.

Each of the above-described assays, however, are limited in theirapplication. Thus, as set forth below and as described in more detail ina co-pending application, filed concurrently herewith and entitled“Relaxin Diagnostic Assays And Kits,” additional assays may also be usedto assay RLF to determine the protein's activity and preferredapplications.

5.4. Production of RLF

RLF may be produced using techniques previously disclosed as useful inproducing relaxin and insulin. For example, the cDNA for RLF disclosedin Burkhardt, et al., 1994, Genomics 20:13-19 and Adham, et al., 1994,J. Biol. Chem. 268:26668-26672 may be used to recombinantly produce RLFaccording to processes previously described as useful in recombinantlymanufacturing relaxin (e.g., U.S. Pat. Nos. 4,758,516, 4,871,670,4,835,251 and U.S. Pat No. 5,464,756 (PCT US90/02085) and PCTUS94/06997. Similarly, such sequence information may be used tosynthesize RLF according to the methods of Bullesbach and Schwabe, 1991,J. Biol. Chem. 266:10754-10761, for synthesizing relaxin.

Derivatives and analogs of RLF also may be synthesized according to themethods of Büllesbach and Schwabe, supra. Alternatively, suchderivatives and analogs may be produced recombinantly using, forexample, site directed mutagenesis techniques as set forth inTsurushita, et al., 1988, Gene 62:135-139.

Relaxin, for use in compositions containing RLF, may be obtained usingany number of readily available techniques.

For example, naturally-occurring relaxin may be purified from a varietyof species including porcine, murine, equine, shark, tiger, rat, dogfishand human. In the human, relaxin is found in most abundance in thecorpora lutea (CL) of pregnancy.

Relaxin may also be synthesized according to the techniques describedabove, with respect to RLF, or alternatively, recombinantly, by relyingupon the disclosed nucleic acid sequences and deduced amino acidsequences for relaxin. In humans, two gene forms encoding for humanrelaxin have been identified, (H1) and (H2) and their use torecombinantly manufacture relaxin, and preferably relaxin (H2), havebeen described. Hudson, et al., 1983, Nature 301 628-631; Hudson, etal., 1984, EMBO J., 3:2333-2339; and U.S. Pat. Nos. 4,758,516 and4,871,670. Methods of making relaxin are also described in U.S. Pat. No.4,835,251 and in U.S. Pat. No. 5,464,756 (PCT US90/02085) and PCTUS94/06997.

Notably, when synthetic human relaxin (H2) and certain human relaxinanalogs were tested for biological activity, the tests revealed arelaxin core necessary for biological activity as well as certain aminoacid substitutions for methionine that did not affect biologicalactivity. Johnston, et al., in Peptides: Structure and Function, Proc.Ninth American Peptide Symposium, Deber, C. M., et al. (eds.) (PierceChem. Co. 1985).

5.5. Indications/Methods of Use

In vitro, proteins having relaxin-like activity decrease collagensynthesis by human dermal and synovial fibroblasts upregulated tooverexpress collagen with transforming growth factor-beta (TGF-beta) orinterleukin-1, and by fibroblasts constitutively overexpressing collagenobtained from scleroderma patients. For example, relaxin decreasescollagen accumulation in vivo in two rodent models of fibrosis. Relaxinor relaxin-like proteins also increase the secretion of thecollagenolytic metalloproteinase, collagenase, and also down-regulatesthe expression of the metalloproteinase inhibitor, tissue inhibitor ofmetalloproteinases.

Relaxin has been implicated consequently in the treatment and diagnosisof various diseases and disorders. For example, studies provide evidencethat relaxin is effective in the treatment of scleroderma, sinusbradycardia, cardiovascular disease, neurodegenerative and neurologicdisorders, hair loss, depression. See e.g., U.S. Pat. No. 5,166,191;U.S. Ser. No. 07/902,637 (PCT US92/069); U.S. Ser. No. 08/483,474 filedconcurrently herewith. Evidence also suggests the use of relaxin indiseases and disorders related to the abnormal expression of collagen orfibronectin, such as scleroderma or rheumatoid arthritis.

As provided herein, RLF possesses relaxin-like biological activity andis therefore similarly implicated in the above described diseases.Moreover, to the extent that RLF is also shown to enhance the activityof relaxin, RLF, as administered in combination with relaxin or anotheragent, is also indicated for the treatment of the above-identifieddiseases.

Additionally, as more fully discussed in the U.S. Application entitled“Relaxin Diagnostic Assays and Kits,” filed concurrently herewith onJun. 7, 1995, U.S. Ser. No. 08/488,399, diagnostic assays fordetermining the predisposition or presence of prostate, breast,testicular, ovarian and other cancers having common stem cell heritage,which rely on detecting the presence of relaxin may also be adjusted torely upon the detection of RLF. Such assays can also be used tofollow-up on tumor metastases after ablation of cancer.

5.6. Pharmaceutical Dosage Requirements, Formulations and Routes ofAdministration

The following dosage requirements, formulations and routes ofadministration for RLF are discussed below:

5.6.1. Effective Dosage.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. More specifically, atherapeutically effective amount means an amount effective to preventdevelopment of or to alleviate the existing symptoms of the subjectbeing treated. Determination of the effective amounts is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes the IC50 asdetermined in cell culture. Such information can be used to moreaccurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms or a prolongation of survivalin a patient. Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g, for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratiobetween LD50 and ED50. Compounds which exhibit high therapeutic indicesare preferred. The data obtained from these cell culture assays andanimal studies can be used in formulating a range of dosage for use inhuman. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See e.g. Finglet al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain therelaxin-like activity and effects.

Administration of RLF, with relain or other active agents, can be viaany of the accepted modes of administration for agents that servesimilar utilities, preferably by systemic administration.

While human dosage levels for treating many of the above-identifiedrelaxin-related diseases or disorders have yet to be optimized for RLF,administered alone or in combination with relaxin, generally, a dailydose is from about 0.1 to 500.0 μg/kg of body weight per day, preferablyabout 6.0 to 200.0 μg/kg, and most preferably about 12.0 to 100.0 μg/kg,depending on whether RLF is administered alone or in combination withrelaxin. Generally it is sought to obtain a serum concentration of RLF,alone or in combination with relaxin, approximating or greater thannormal circulating levels in pregnancy, i.e., 1.0 ng/ml, such as 1.0 to20 ng/ml, preferably 1.0 to 20 ng/ml.

For administration to a 70 kg person, the dosage range would be about7.0 μg to 3.5 mg per day, preferably about 42.0 μg to 2.1 mg per day,and most preferably about 84.0 to 700.0 μg per day. The amount of RLFadministered will, of course, be dependent on the subject and theseverity of the affliction, the manner and schedule of administrationand the judgment of the prescribing physician. One treatment regimen canemploy a higher initial dosage level (e.g., 100 to 200 μg/kg/day)followed by decreasing dosages to achieve steady relaxin or relaxin-likeserum concentration of about 1.0 ng/ml. Another treatment regimen,particularly postpartum depression, entails administration of an amountof relaxin sufficient to attain normal pregnancy levels of relaxin(about 1.0 ng/ml) followed by gradual decreasing dosages until relaxinserum levels are no longer detectable (e.g. less than about 20picograms/ml), optionally discontinuing treatment upon reaching thatdosage level.

In employing RLF, either alone or in combination with relaxin, fortreatment of the above conditions, any pharmaceutically acceptable modeof administration can be used. RLF can be administered either alone orin combination with other pharmaceutically acceptable excipients,including solid, semi-solid, liquid or aerosol dosage forms, such as,for example, tablets, capsules, powders, liquids, gels, suspensions,suppositories, aerosols or the like. Relaxin can also be administered insustained or controlled release dosage forms (e.g., employing a slowrelease bioerodable delivery system), including depot injections,osmotic pumps (such as the Alzet implant made by Alza), pills,transdermal (including electrotransport) patches, and the like, forprolonged administration at a predetermined rate, preferably in unitdosage forms suitable for single administration of precise dosages. Thecompositions will typically include a conventional pharmaceuticalcarrier or excipient and RLF. In addition, these compositions mayinclude other active agents, carriers, adjuvants, etc.

In a preferred aspect of the invention, a sustained/controlled releaseRLF formulation was a selectively permeable outer barrier with a drugdispensing opening, and an inner RLF-containing portion designed todeliver dosage of RLF progressively diminished as a predetermined rate(e.g. containing about 30 mg of RLF in a matrix for delivery ofinitially about 500 μg per day diminishing as a rate of 10 μg per day.

In another preferred aspect of the invention, a sustained/controlledrelease RLF formulation has a selectively permeable outer barrier with adrug dispensing opening, a first inner relaxin-containing portiondesigned for steady state release of relaxin at a therapeuticallyeffective daily dosage (e.g. containing about 50 mg of relaxin in amatrix for continuous delivery of about 500 μg per day), and a secondinner RLF-containing portion designed to deliver a dosage of RLFprogressively diminishing at a predetermined rate (e.g. containing about3 mg of relaxin in a matrix for delivery of initially about 500 μg perday diminishing at a rate of 50 μg per day) commencing upon exhaustionof the relaxin from the first inner portion.

Generally, depending on the intended mode of administration, thepharmaceutically acceptable composition will contain about 0.1% to 90%,preferably about 0.5% to 50%, by weight of RLF, either alone or incombination with relaxin, the remainder being suitable pharmaceuticalexcipients, carriers, etc. Actual methods of preparing such dosage formsare known, or will be apparent, to those skilled in this art; forexample, see Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa., 15th Edition, 1975. The formulations of humanrelaxin described in U.S. Ser. No. 08/050,745 are particularlypreferred.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

5.6.2. Routes of Administration.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, or intestinal administration. Parenteraladministration is generally characterized by injection, eithersubcutaneously, intradermally, intramuscularly or intravenously,preferably subcutaneously. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol or the like. In addition, if desired, thepharmaceutical compositions to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, solubility enhancers, and the like, such asfor example, sodium acetate, sorbitan monolaurate, triethanolamineoleate, cyclodextrins, and the like.

The percentage of RLF and/or relaxin contained in such parenteralcompositions is highly dependent on the specific nature thereof, as wellas the needs of the subject. However, percentages of active ingredientof 0.01% to 10% in solution are employable, and will be higher if thecomposition is a solid which will be subsequently diluted to the abovepercentages. Preferably the composition will comprise 0.2-2% of the RLF,alone or in combination with relaxin in solution.

A more recently devised approach for parenteral administration employsthe implantation of a slow-release or sustained-release system, suchthat a constant level of dosage is maintained. See, e.g., U.S. Pat. No.3,710,795.

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a solid tumor, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with tissue-specific antibody.The liposomes will be targeted to and taken up selectively by thetissue.

5.6.3. Composition/Formulation.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

A pharmaceutical carrier for the hydrophobic compounds of the inventionis a cosolvent system comprising benzyl alcohol, a nonpolar surfactant,a water-miscible organic polymer, and an aqueous phase. The cosolventsystem may be the VPD co-solvent system. VPD is a solution of 3% w/vbenzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5%dextrose in water solution. This co-solvent system dissolves hydrophobiccompounds well, and itself produces low toxicity upon systemicadministration. Naturally, the proportions of a co-solvent system may bevaried considerably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied: for example, other low-toxicity nonpolar surfactants maybe used instead of polysorbate 80; the fraction size of polyethyleneglycol may be varied; other biocompatible polymers may replacepolyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars orpolysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are well knownexamples of delivery vehicles or carriers for hydrophobic drugs. Certainorganic solvents such as dimethylsulfoxide also may be employed,although usually at the cost of greater toxicity. Additionally, thecompounds may be delivered using a sustained-release system, such assemipermeable matrices of solid hydrophobic polymers containing thetherapeutic agent. Various of sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Formulations of RLF may also be administered to the respiratory tract asa nasal or pulmonary inhalation aerosol or solution for a nebulizer, oras a microfine powder for insufflation, alone or in combination with aninert carrier such as lactose, or with other pharmaceutically acceptableexcipients. In such a case, the particles of the formulation mayadvantageously have diameters of less than 50 microns, preferably lessthan 10 microns. See, e.g., U.S. Pat. No. 5,364,838, which discloses amethod of administration for insulin that can be adapted for theadministration of RLF, alone or in combination with relaxin in thepresent invention.

RLF for treatment of such disorders such as alopecia, may also beadministered topically in a formulation adapted for application to thescalp, such as a shampoo (e.g., as disclosed in U.S. Pat. No. 4,938,953,adapted according to methods known by those skilled in the art, asnecessary for the inclusion of protein ingredients) or a gel (e.g., asdisclosed in U.S. Pat. No. 5,451,572) optionally with increased relaxinconcentrations to facilitate absorption.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

5.6.4. Packaging

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. Compositions comprisinga compound of the invention formulated in a compatible pharmaceuticalcarrier may also be prepared, placed in an appropriate container, andlabelled for treatment of an indicated condition. Suitable conditionsindicated on the label may include treatment of depression, sinusbradycardia, hair loss, neurologic or neurodegenerative diseases,scleroderma, cardiovascular disease or disorders or diseases related touncontrolled or abnormal collagen or fibronectin formation.

More specific dosage, formulation and methods of administration may bederived from information contained in U.S. Pat. No. 5,166,191, PCTUS92/06927 and U.S. Pat.No. 5,451,572 and co-pending applications, filedconcurrently herewitht.

6. EXAMPLES

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

6.1. RLF Synthesis and Confirmation of Synthesized Protein

As described above, RLF may be produced by isolating the protein fromnatural sources, synthesizing the protein based on RLF's deduced aminoacid sequence and recombinantly manufacturing the protein based uponavailable cDNA data.

One procedure for synthesizing RLF is as follows:

Materials. L-Amino acid derivatives for peptide synthesis were purchasedeither from Bachem Bioscience (Philadelphia, Pa.) or Bachem Calif.(Torrance, Calif.). Solvents for peptide synthesis and chromatographywere distilled in glass (Burdick and Jackson; Muscagon, Mich.), and thechemicals for peptide synthesis were obtained from Perkin Elmer AppliedBiosystems (Foster City, Calif.). Other chemicals of analytical gradewere used without further purification.

Methods. The following method was followed to synthesize RLF:

Peptide Synthesis: The B chain of the RLF protein was synthesized bytert.butyloxycarbonyl¹-chemistry using conventional HF-labile sidechain-protecting groups for all three functional amino acids exceptcysteines. Cysteine B10 was protected by the acetamidomethyl group andB23 by the thiol-protecting/activating group[S-(3-nitro-2-pyridinesulfenyl)](CysB23). Methionine was protected bysulfoxide formation, and tryptophan by the N(in)formyl group. Thesynthesis was performed on an Applied Biosystems peptide synthesizermodel 430A on [4-(oxymethylphenylacetamidomethyl]resin loaded with 0.4mmol tert butyloxycarbonyl-alanine. Deprotection and removal from thesolid support was accomplished by HF-treatment in the presence of 5%m-cresol. The crude peptide was extracted with 20% acetic acid andlyophilized (yield 1.387 g). The B chain was purified on Sephadex G50-sf(2.5 cm×50 cm) in 1 M acetic acid (yield: 840 mg), followed bypreparative HPLC on Synchropak RP-P (2.1 cm×25 cm) in portions of 50 to70 mg. The mobile phase consisted of 0.1% trifluoroacetic acid (TFA) inwater (solvent A) and 0.1% TFA in 80% acetonitrile (solvent B). Thecolumn was equilibrated in 20% B and the peptide eluted with a lineargradient of 20% B to 50% B over 1 h at a flow rate of 5 ml/min (overallyield: 233 mg). Amino acid composition: Thr 2.00 (2); Ser 0.86 (1); Glu2.90 (3); Gly 3.28 (3); Ala 2.16 (2); Cys 0.89 (2); Val 3.19(3); Met1.22 (1); Leu 1.94 (2); Phe 0.99 (1); His 2.44 (2); Lys 0.96 (1); Arg3.81 (4).

The A chain (0.25 mmol) was synthesized via Fast-moc chemistry on an ABIpeptide synthesizer (model 430A) on p-benzyloxybenzyl resin. All sidechains were protected by TFA-abile protecting groups except Cys(A11),which was acetamidomethyl-protected, and Cys (A24) which was protectedby the HF-labile p-menthylbenzyl group. The A chain was deprotected withTFA/thiophenol (10:1 v/v), using 50 mg peptidyl resin/ml for 90 min atroom temperature (5). The TFA was evaporated and the peptideprecipitated with ether. The precipitate was collected bycentrifugation, the supernatant discarded, and the pellet washed twicewith ether and air-dried. The peptide was suspended in water, dissolvedby the addition of ammonia, and desalted on Sephadex G25-m in 50 MMNH₄HCO₃. To the eluate (100 ml) 50 ml of Me₂SO was added in order toaccelerate the oxidation of the intrachain disulfide bond A10-A15 (6).The progress of oxidation was observed by the Ellman reaction (7). Aftercompletion of the disulfide bond formation the A chain was dialyzedagainst water and lyophilized (yield 372.3 mg). Aliquots of 20 mg werefurther purified by preparative HPLC on Synchropak RP-P (10 mm×250 mm),using 0.1% TFA in water for solvent A and 0.1% TFA in 80% acetonitrilefor solvent B. The column was equilibrated in 30% B and the peptideeluted with a linear gradient of 30% B to 50% B over 30 min at a flowrate of 3 ml/min (overall yield: 166.5 mg). Amino acid composition: Asp2.20 (2); Thr 3.00 (3); Ser 0.99 (1); Glu 1.92 (2); Pro 2.25 (2); Gly1.06 (1); Ala 4.18 (4); Cys 1.62 (4); Leu 3.60 (4); Tyr 1.82 (2); Arg0.98 (1).

For chain combination, 33.4 mg (11.3 μmol) of the Achain(acetamidomethylA10, 4-methylbenzylA24) was treated with 4 ml of HFin the presence of 200 μl of m-cresol for 45 min at 0° C. Thereafter theHF was evaporated in a stream of nitrogen and the peptide precipitatedwith ether. The pellet was collected and dried over KOH in vacuo for 30min. The monothiol A chain was dissolved in 4 ml of 8 Mguanidiniumchloride in 0.1 M acetic acid at pH 4.5 and added to 36.3 mg(9.6 μmol) of the B chain. The disulfide bond A24/B23 was formed at 37°C. for 24 h and the resulting product separated first onSephadex G50-sfin 1 M acetic acid (column 2.5 cm×50 cm) (yield 48.7 mg, 78.3%),followed by preparative HPLC on Synchropak RP-P (10 mm×250 mm) using0.1% TFA in water for solvent A and 0.1% TFA in 80% acetonitrile forsolvent B. The column was equilibrated in 30% B and the peptide elutedwith a linear gradient of 30% to 45% B over 30 min at a flow rate of 3ml.min (yield: 34.1 mg, 54.8%).

The resulting peptide contained acetamidomethyl groups in positions CysA11 and Cys B10, the N(in) formyl group in Trp B27, and a sulfoxide inthe side chain of Met B5. For the formation of the third disulfide bondthe peptide (9.3 mg) was dissolved in water (3.5 ml) and added to astirred solution consisting of acetic acid (3.5 ml) 6 N HCl (19.1 μl)and 3 ml of 50 mM iodine in acetic acid (8). The reaction was performedat room temperature for 10 min, quenched with ascorbic acid, and theproduct was desalted on Sephadex G25-sf in 1 M acetic acid andlyophilized. After purification by preparative HPLC (conditions asbefore) (yield: 3.42 mg, 36.8%) the protein still contained protectinggroups in Trp (B27) and Met (B5).

Complete deprotection was achieved first by treatment of 11.3 mg of thepeptide with 2 ml of water/piperidine 9:1 (v/v) for 2 min at roomtemperature. The base was neutralized with 0.4 ml acetic acid and thepeptide purified by preparastive HPLC, dried (yield:11.0 mg, 97.5%), and10 mg of peptide-containing methionine sulfoxide was reduced with 1 mlof TFA/0.5 M NH₄I in water 9:1 v/v for 15 min at 0° C. Free iodine wasreduced with 0.5 M ascorbic acid in water and the reaction quenched bydilution with water. The final peptide was recovered by preparative HPLC(conditions as before) (yield 7.57 mg=75.7%). Amino acid compositions:Asp 2.02 (2), Thr 4.79 (5), Ser 1.77 (2), Glu 4.86 (5), Pro 5.17 (5),Gly 4.15 (4), Ala 6.09 (6), Cys 3.51 (6), Val 2.86 (3), Met 0.70 (1),Ile 0 (0), Leu 5.74 (6), Tyr 2.12 (2), Phe 0.98 (1), His 2.00 (2), Lys1.26 (1), Trp 1.00 (1), Arg 5.02 (5). (overall yield 14.9%).

The mobile phase of all HPLC systems used consisted of 0.1%trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic acidin 80% acetonitrile (solvent B).

For preparative HPLC, a Waters HPLC system consisting of two pumps(model 6000A) and gradient programmer (model 680) was used incombination with a Synchropak RP-P column (C18) (SynChrom, In) and anUvicord S UV (226 nm) monitor (LKB, Bromma Sweden). Usually 1 to 20 mgof peptide was separated using linear gradients as indicated.

Analytical HPLC I was performed on Aquapore 300 (C₈;2.1 mm×30 mm) usingan Applied Biosystems HPLC model 130A. Separation was achieved with alinear gradient from 23% to 34% B in 60 min at a flow rate of 0.1%ml/min. The peptide was detected by UV absorbance at 230 nm.

Analytical HPLC II was performed on Synchropak RP-P (C18, 4.1 mm×250 mm)using a Waters HPLC system. Separation was achieved with a lineargradient from 20% B to 50% in 30 min at a slow rate of 1 ml/min. Thepeptide was detected by UV absorbance at 220 nm. The above-describedHPLC may also be used to verify the purity of the RLF, as set forth inFIG. 3.

The primary structure of RLF, as synthesized according to the aboveprocedure, or any other solid phase methodologies in combination withsite-directed sequential disulfide bond formation (a schematic depictingsaid formation is set forth at FIG. 2), is set forth at FIG. 1.

Protein Confirmation and Verification. The identity of the synthesizedRLF may be confirmed and verified according to known techniques.

Amino Acid Analyses: Following protein synthesis and purification, aminoacid analyses was conducted to confirm the protein's identity. First,peptides were hydrolyzed in vapor phase 6N HCI containing 0.1% phenolfor 1 h at 150° C. The amino acids were detected after pre-columnmodification with phenylisothiocynate and separation by HPLC (Pico•Tagsystem, Waters Millipore).

Sequence Analyses: The identity of the protein was verified by sequenceanalysis. Specifically, such analyses were performed on an ABI 477 Apulsed liquid protein sequencer and an in-line ABI 120Aphenylthiohydantoin analyzer (ABI, Applied Biosystems, Foster City,Calif.). Chains were prepared by reduction of about 10 μl of therelaxin-like factor in 20 μl 50 mM DTT in 3 M guanidinium chloride, 0.2M Tris, HCI at pH 8.5 for 1 h at 37° C., diluted with 30 μl of solventA, followed by separation on Aquapore 300 (see: Analytical HPLC forconditions).

Upon reduction two chains were generated, isolated, and the subsequentsequence analyses of both chains showed the desired structure.

UV Spectroscopy: The confirmation and structure of the synthesizedprotein was then confirmed by UV spectroscopy. Such spectroscopy wasperformed on an OLIS Cary 15 spectrophotometer conversion (On-LineInstrument Systems Inc., Bogart, Ga.). UV-spectroscopy was used todetermine the protein concentrations of the RLF protein. The specificabsorption coefficient (ε₂₇₆=1.40 cm₂/mg) was obtained by directcomparison of UV absorbance and the recovery of amino acids afterhydrolysis and amino acid analysis.

No partial hydrolysis at the acid labile Asn-Pro bond was detected.

Circular Dichroism: Further confirmation of the synthesized protein'sidentity was performed by CD spectroscopy, as performed on a Jasco J-710spectropolarimeter using a cell of 0.02 cm path length. Proteins weredissolved in 25 mM Tris/HCI at pH 7.7 and concentrations were determinedby UV spectroscopy: 0.67 mg/ml for porcine relaxin, 0.54 mg/ml forrelaxin-like factor, and 0.55 mg/ml for human relaxin. Spectra weremeasured at a resolution of 0.2 nm, a band width of 2 nm, and 5 spectrawere averaged. Molar ellipticity was calculated according to Adler etal. (9) using mean residual weights of 110.4 for relaxin-like factor,113.6 for porcine relaxin, and 112.5 for human relaxin.

Comparative measurements of circular dichroic spectra suggested nearidentity of the solution structures of RLF and porcine relaxin. See,FIG. 4.

Mass Spectrometry: Finally, mass spectra were recorded on a JEOLHX110/HX110 4 sector tandem mass spectrometer (JEOL, Tokyo, Japan) toverify the protein's identity and proper synthesis. Samples weredissolved in 0.1% trifluoroacetic acid at a concentration of about 0.8nmol/μl.

Mass spectrometry showed the correct mass ion for the synthetic RLF(found: 6294.6, theoretical 6293.2).

6.2. Production of Labelled RLF

¹²⁵I-labeled RLF, containing side chain-protected tryptophan andmethionine, may be prepared according to the above procedure wherein thesynthesized peptide (10 μg in 5 μl of water) is then placed into a 200μl Eppendorf vial and 5 μl phosphate buffer (250 mM, pH 7.4), followedby 2 μl of ¹²⁵I-(1 mCi), and 5 μl of chloramine T (2 mg/ml in phosphatebuffer pH 7.4) are added. The reaction was performed for 1 min on ice,quenched by addition of 5 μl of sodium thiosulfate (5 H₂O) (50 mg/ml inphosphate buffer pH 7.4), and 5 μl of NaI (20 mg/ml in phosphate bufferpH 7.4). The side chain-protecting group of Trp was removed by additionof 5 μl of piperidine. After 2 min at room temperature the reaction wasquenched by the addition of 5 μl of glacial acid, the reaction mixturewas diluted with 10 μl of water and loaded onto a Aquapore 300 columnfor separation. The protein was detected by UV absorbance and peaks weremanually collected into 100 μl of 1% bovine serum albumin in water.

The labelled RLF may be used as an RLF tracer which could then be usedto separate by HPLC the different RLF derivatives to yield acarrier-free tracer. See, FIG. 5. Alternatively, such labelled RLF mayalso be used in binding assays and for RLF receptor mapping.

6.3. Receptor Binding Assay.

Insulin-receptor binding assays were performed on crude membranepreparations of term placenta, as described in Hock and Hollenberg,1980, J. Biol. Chem. 255:10731-10736, using ¹²⁵I-iodo-Tyr^(A14) porcineinsulin as tracer according to the method of Linde, et al., 1986, J.Chromatogr. 369:327-339.

The assays were performed in HMS-buffer (25 mM HEPES, 104 mM NaCl, 5 mMMgCl₂, 0.2% bovine serum albumin; pH 7.4) in a total volume of 100 μl.Labeled insulin (50,000 cpm/assay, 150 pM) and variable amounts ofinsulin were incubated with crude membranes for 1 h at room temperature.Thereafter 1 ml of buffer was added, the membranes collected bycentrifugation in a microcentrifuge at 14,000 rpm for 5 min, thesupernatant discarded, and the tip of the Eppendorf vial cut off andcounted in a γ-counter (Minigamma, LKB, Sweden). To determinenonspecific binding unlabeled insulin was used at a concentration of 2μl/ml (0.33 μM) and nonspecific binding was usually below 10% of thetotal binding.

Contrary to prior art speculation, RLF does not bind with anysignificant degree to the insulin receptor.

6.4. Relaxin-Binding Assays

Relaxin-binding assays were performed as described in Yang, et al.,1992, Endocrinology 130:179-185 and Büllesbach, et al., 1994, Endocrine.2:1115-1120, using crude membrane preparations of mouse tissue. Mousebrains of 2 mice were collected into 15 ml of chilled buffer (25 mMHEPES, 0.14 M NaCl, 5.7 mM KCI, 0.2 mM phenylmethylsulfonyl-fluoride,and 80 mg/ml soybean trypsin inhibitor, pH 7.5) supplemented withsucrose (0.25 M, final concentration). The tissue was homogenized on icefor 10 s with a Polytron homogenizer (Brinlmann, Westbury, N.Y.) atsetting 5. The homogenate was centrifuged at 700 rpm for 10 min at 4° C.and the supernatants were recentrifuged at 10,000×g for 1 h. The pelletwas resuspended in 15 ml of ice cold binding buffer, 25 mM HEPES, 0.14 MNaCl, 5.7 mM KCI, 0.2 mM phenylmethylsulfonylfluoride, and 80 mg/mlsoybean trypsin inhibitor, pH 7.5, supplemented with 1% bovine serumalbumin, and centrifuged for 1 h at 10,000×g. The crude membranepreparation was suspended in 1 ml of binding buffer and 40 μl was usedper assay. The assay was performed using 40 μl of tracer (about 100,000cpm of porcine relaxin tracer=150 pM) and 20 μl of relaxin at variousconcentrations. The assay was incubated for 1 h at room temperature, andthe suspension diluted with 1 ml of wash buffer (25 mM HEPES, 0.14 MNaCl, 5.7 mM KCI, 1% bovine serum albumin, 0.01% NaN₃) and centrifugedin Eppendorf centrifuge at 14,000 rpm for 10 min. The supernatant wasdiscarded, the tip of the vial cut and counted in a γ-counter.Nonspecific binding was determined in the presence of 2 μl/ml ofunlabeled competitor (0.33 μM). In a typical experiment the specificbinding was between 25% and 40% of the total binding.

Tissue specificity was determined using crude membrane preparations ofleg muscles, kidneys, liver. brain, and uterus (of estrogen primedmice). The crude membranes were prepared as described for relaxin.Binding was based on protein concentration determined by Lowry.

According to the above-described assay, a hundred-fold excess of humanRLF displaces 50% of the relaxin tracer from a mouse brain relaxinreceptor preparation. The difference in affinity is still within therange of specific binding, i.e., several orders of magnitude better thanthe binding of insulin or guinea pig relaxin to this receptor (seegenerally, Büllesbach, et al., 1994, Endocrine. 2:1115-1120) indicatingthat RLF recognizes the relaxin receptor.

As discussed above, this result was surprising because the criticalsequence in RLF consisting of two Ara residues separated by three aminoacids is offset toward the C-terminal end of the B chain by exactly oneturn of the helix (See, FIG. 1).

Of the tissues tested with ¹²⁵I-RLF as tracer such as brain, uterus,skeletal muscle, kidney and liver, only the brain- and the uterusmembrane preparation showed specific binding (See, FIG. 6). These aretissues that also bind relaxin in a competitive and saturable manner. Totest for crossreactivity the assays were performed with tracers andcompetitive cold molecules exchanged. The results from such assay areset forth below at Table 1:

TABLE 1 Relaxin-Binding Assay Results Tissue 50% (mouse) TracerCompetitor Binding Range Brain pRLX hRLX 1 ng 0.1-2.0 Brain pRLX hRLF200 ng 190-220 Uterus pRLX hRLX 1 ng 0.1-2   Uterus pRLX hRLF 10000 ng —Uterus hRLF hRLX 800 ng  600-1000 Uterus hRLF hRLF 0.3 ng 0.1-0.6 BrainhRLF hRLX 1000 ng  900-1100 Brain hRLF hRLF 0.3 ng 0.1-0.6 pRLX =porcine relaxin, hRLX = human relaxin, pRLF = porcine RLF and hRLF =human RLFThese results suggest strongly that RLF does have its own receptor inthese tissues and that the relaxin receptor is recognized by RLF, butwith a significantly lower affinity than relaxin. Furthermore, the datasupport that the brain and uterine relaxin receptors differ with respectto crossreactivity. Specifically, the uterine relaxin receptor barelyrecognizes RLF whereas the brain receptor shows moderatecrossreactivity. In general, the RLF receptor binds its substrate withgreater affinity than the relaxin receptor displays toward relaxin.

6.5. Sperm Motility Assay

Relaxin and proteins having relaxin-like activity may be identified by asperm motility assay.

Materials And Methods. Semen is obtained by masturbation from healthyvolunteers. The sample is allowed to liquefy at room temperature and isthen mixed with Minimum Essential Medium (MEM) with Hepes buffer added.This medium is used because it coincides with that washing mediumemployed by the in vitro clinic at MUSC. The sperm is then separatedfrom the seminal fluid and MEM by centrifugation. The resultant spermpellet is then resuspended in MEM at room temperature. Aliquots are thenplace in siliconized centrifuge tubes and one of several compoundsadded: l)human relaxin 10 ng/ml, 2)human relaxin 100 ng/ml,3)relaxin-like factor 10 ng/ml, 4)relaxin-like factor 100 ng/ml, 5)onefraction of alkaline gland fluid from stingrays diluted 1:8 withpentoxyfyline. The additive is mixed well with the sperm/medium mixture.Samples are taken at 0, 2, 4, 6 and 24 hour intervals for automateddetermination of the following parameters: 1)motility, 2)progressivity,3)path velocity, 4)progressive velocity, 5)track speed, 6)elongation,7)lateral displacement, 8)cross beat frequency, 9)straightness,10)linearity. Briefly described, each sample is loaded into a Makerheated specimen chamber and viewed in a light microscope equipped withlaser doppler optics (IVOS, Beverly, Mass.). Sample readings taken atapproximately 3 minutes and results are displayed in hard copy form.

Experimental Results. Human relaxin and RLF added to washed human spermpreserve the motility compared to untreated controls in which motilityand thus potential fertilizing capacity significantly declines overtime. There was essentially no difference between the high and low dosesof relaxin in their effects on motility. RLF was as potent inmaintaining sperm motility at both doses as was relaxin. The moststriking effect of both compounds occurred at 4 hours when motilityremained the same or increased from the previous time period.

Both relaxin and relaxin-like factor were given in combination todetermine if there was an additive effect. No such effect was observed.

All the above compounds were compared against stingray alkaline glandfluid (ALG) from the HPLC. AGF significantly increased then maintainedsperm motility for the first two time periods then was approximately 10%higher for the last three time periods.

6.6. In Vitro Inhibition of Collagen and Fibronectin Expression by HumanLung Fibroblasts

Whether RLF inhibits collagen and fibronectin expression has beenstudied in the context of human lung fibroblasts. Specifically, RLF(1-100 ng/ml) was applied to human lung fibroblasts in serum-free mediumand assayed for collagen secretion by biosynthetic labelling with₃H-proline in the presence of ascorbate and B-aminopropionitrile. Whentested on lung fibroblasts stimulated with TGF-β, RLF's ability toinhibit collagen expression at various dose levels can be determined.The presence of another extracellular matrix molecule, fibronectin, inconditioned media was assessed by Western immunoblotting using ananti-fibronectin polyclonal antibody, as well as biosynthetic labeling.

6.7. In Vitro Inhibition of Collagen and Fibronectin Expression bySynovial Fibroblasts

Trauma to the shoulder or surgical intervention of large joints is oftenassociated with limitation in mobility, in many cases due to anexaggerated fibrotic response to synovial or capsular tissue.Extracellular matrix-producing cells, such as synovial fibroblasts, arecapable of the extremes of degradation or repair. The overproduction ofcollagens, fibronectin and other extracellular matrix molecules can bedue to the local expression of cytokines, such as transforming growthfactor TGF-β. To the extent it has been demonstrated that relaxin candecrease TGF-β-stimulated collagen expression in a dose-dependentmanner, up to 30% at a relaxin does of 100 ng/ml and fibronectinexpression by 30%, RLF is also implicated in the modulation and controlof collagen and fibronectin expression.

To test such hypothesis (to determine RLF's ability to down-regulatecollagen and fibronectin expressed by synovial fibroblasts), fibroblastscan be explanted from pieces of rheumatoid synovium and treated withTGF-beta (1 ng/ml) to stimulate expression of types I and III collagens.TGF-beta upregulated collagen expression at the protein level, asmeasured by biosynthetic labelling with 3H-proline incorporation. Morespecifically, the following experiments were conducted to determineRLF's ability to modulate the expression of collagen, fibronectin andprocollegenase in human synovial fibroblasts:

6.7.1. Assay to Determine the Inhibition of Collagen Expression

The method for detecting and measuring collagen formation in thepresence of relaxin described in Unemori and Amento, 1990, J. Biol.Chem. 265:10681-685 has been modified as follows to determine theability of RLF, in vitro, to modulate the expression of collagen.

Materials and Methods. Rheumatoid synovial fibroblasts (Strain No.RSF64) were seeded at a density of 6.25×10⁴ cells/cm² in tissue culturedishes in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with10% fetal bovine serum. After 24 hours, the cells were washed andtreated with DMEM supplemented with 0.2% lactalbumin hydroxylsate withrelaxin, RLF and/or transforming growth factor (TGF-β).

The cells were simultaneously biosynthetically labelled with ³H=proline(25 μCi/ml) in the presence of ascorbate and BAPN. After 24 hours, theconditioned media were collected and electrophoresed on 4-12%polyacrylamide gels (NOVEX) under reducing conditions. Gels wereenhanced, dried, and exposed to X-ray films for 1-2 weeks. Collagenbands were identified on the X-ray films as bacterialcollagenase-sensitive, proline-incorporating bands between 95-200 kDa.Band density was quantified by scanning densitometry and used asestimates of collagen expression.

Experimental Results. Using the above protocol, it was determined thatRLF decreases collagen expression independently. Specifically,fibroblast treatment with TGF-β increased collagen expression by 3.75fold over that expressed by untreated fibroblasts. Subsequent additionof RLF (100 ng/ml) to the TGF-β-treated fibroblasts decreasedTGF-β-stimulated collagen expression by 17%. In comparison, addition ofrelaxin (100 ng/ml) decreased TGF-β-stimulated collagen expression by9%.

It was further determined that RLF and relaxin together synergisticallydecrease collagen expression. Specifically, treatment ofTGF-β-stimulated cells with RLF (100 ng/ml) and relaxin (100 ng/ml)decreased collagen expression by 39%.

6.7.2. Assay to Determine the Inhibition of Fibronectin Expression

The method for detecting and measuring collagen formation in thepresence of relaxin described in Unemori and Amento, 1990, J. Biol.Chem. 265:10681-685 has been modified as follows to determine theability of RLF, in vitro, to modulate the expression of fibronectin.

Materials And Methods. Specifically, using the method described insection 6.7.1., the fibronectin band was identified by size (220 kDa),bacterial collagenase-resistance, and positive staining usingcommercially available polyclonal anti-fibronectin antibody (Promega).The fibronectin band was scanned densitometrically to estimate levels ofexpression.

Experimental Results. Using the above protocol, it was determined thatRLF decreases fibronectin expression independently. Specifically,addition of RLF (100 ng/ml) to the TGF-β-treated fibroblasts decreasedTGF-β-stimulated fibronectin expression by 17%.

6.8. In Vitro Stimulation of Procollagense Expression by SynovialFibroblasts

A method for detecting and measuring procollagenase formation isdescribed in Unemori, et al., 1991, J. Biol. Chem. 266:23477-482. Suchmethod was modified to measure the expression of procollagenase in thepresence of RLF as follows:

Materials And Methods. Rheumatoid synovial fibroblasts (Strain No.RSF112) were seeded at a density of 6.25×10⁴ cells/cm² in tissue culturedishes in DMEM supplemented with 10% fetal bovine serum. Aftertwenty-four hours, the cells were washed and treated with DMEMsupplemented with 0.2% lactalbumin hydroxylate with relaxin at 1, 10 and100 ng/ml for 48 to 72 hours. Conditioned media were collected and analiquot analyzed by gelatin zymography. Procollagenase was identified asa gelatinlytic doublet at 52/57 kDa. The intensity of the doublet (i.e.,the amount of procollagenase expressed, was quantified by scanningdensitometry.

Experimental Results. RLF stimulated expression of procollagenase in adose-dependent manner comparable to that induced by relaxin. RLF at 1,10 and 100 ng/ml stimulated procolleganse expression by 0, 2.0 and4.2-fold. Relaxin induced procollagenase expression by 0, 1.6 and4.9-fold at the equivalent doses.

6.9. Cyclic AMP-Release Bioassay

The cAMP assay is a competitive immunoassay commercially availablethrough Amersham Corporation.

Materials and Methods. To determine cAMP release induced by RLF, normalhuman endometrial cells are grown at 1.2×10⁴ cells/well in a 96-wellplate in DMEM/F12+10% newborn calf serum. 24 hours later, the cells arewashed in serum-free medium comprised of DMEM/F12+0.2% lactalbuminhydrolysate. 24 hours later, the cells are treated with relaxin and/orRLF in the presence of isobutylmethylxanthine and forskolin for 30 min.The cell lysates are harvested with 0.1N HCL, neutralized with 0.1NNaOH, then assayed in the immunoassay (Amersham Corp).

Experimental Results. When relaxin was assayed at 0.78 ng/ml, 86 pM cAMPwas measured in endometrial cell lysates. When RLF (2.5 ug/ml) wassimultaneously added, 470 pM cAMP, roughly a 5-fold enhancement in cAMPproduction, was measured. When a relaxin concentration of 3.12 ng/ml wastested with and without RLF (2.5 ug/ml), a 2-fold enhancement wasmeasurable with relaxin+RLF as compared with relaxin alone.

6.10. Mouse Symphysis Pubis Assay

Mouse interpubic ligament assays were performed essentially as describedby Steinetz, et al., 1960, Endocrinology 67:102-115. Ovariectomizedvirgin female mice were printed with 5 μg estrogen cypionate in 100 μlsesame oil. Five days later the mice were injected subcutaneously withhuman relaxin, RLF, or mixtures of human relaxin and RLF in 100 μl of0.1% benzopurpurin 4B. Specifically, groups of five animals receivedeither human relaxin at a suboptimal dose or a mixture of 0.2 μg, 0.4 μgand 0.8 μg human relaxin and 5 μg of RLF as given in FIG. 8. Fornegative control 100 μl of 0.1% benzopurpurin 4B in water were injected.After 16 hours the mice were killed in an atmosphere of CO₂, thesymphysis pubis dissected free, and the distance between the interpubicbones measured with a dissecting microscope fitted withtransilluminating fiber optics.

The RLF significantly increased the activity of human relaxin in themouse bioassay. Increasing RLF concentrations in the presence of 0.5 μgof human relaxin showed that 5 μg of RLF was optimal (FIG. 9). Again theeffect of the RLF is clearly recognized. In the next assay relaxinalone. RLF alone, and a maximal dose of both are compared (FIG. 7).While RLF alone had no effect, the relaxin effect at maximal dose wasstill augmented by RLF.

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention, and methods which are functionally equivalent are within thescope of the invention. Indeed, various modifications of the inventionin addition to those described herein will become apparent to thoseskilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

All references cited within the body of the instant specification arehereby incorporated by reference in their entirety. In addition, thepublications listed below are of interest in connection with variousaspects of the invention and are incorporated herein as part of thedisclosure:

1. Adham, et al., 1993, J. Biol. Chem. 268:26668-26672;

2. Adler, et al., 1973, Methods Enzymol. 27:675-735;

3. Büllesbach, et al., 1994, Endocrine. 2:1115-1120;

4. Büllesbach and Schwabe, 1994, J. Biol. Chem. 269:13124-13128;

5. Büllesbach and Schwabe, 1993, Biochem. Biophys. Res. Commun.196:311-319;

6. Büllesbach and Schwabe, 1992, J. Biol. Chem. 267:22957-22960;

7. Büllesbach and Shcwabe, 1991, J. Biol. Chem. 266:10754-10761;

8. Büllesbach, et al., 1980, Hoppe Seyler's Z. Physiol. Chem.361:865-873;

9. Burkhardt, et al., 1994, Genomics 20:13-19;

10. Eddie et al., 1986, Lancet 1:1344-1346;

11. Eigenbrot, et al., 1991, J. Mol. Biol. 221:15-21;

12. Ellman, 1959, Arch. Biochem. Biophys. 82:70-7.7;

13. Hock and Hollenberg, 1980, J. Biol. Chem. 255:10731-10736;

14. Linde, et al., 1986, J. Chromatogr. 369:327-339;

15. Loumaye et al., 1978, Gynecologic and Obsteric Investigation9:262-267;

16. Olefsky, et al., 1982, Ann. NY Acad. Sci. 380:200-216;

17. Rembiesa, et al., 1993, Endocrine J. 1:263-268;

18. Schwabe and Büllesbach, 1994, FASEB J. 8:1-2;

19. Schwabe and Harmon, 1978, Biochem. Biophys. Res. Commun. 84:374-380;

20. Sherwood et al., 1980, Endocrinology 107:691-698;

21. Sherwood and Crnekovic, 1979, Endocrinology 104:893-897;

22. Sieber, et al., 1977, Helv. Chim. Acta 60:27-37;

23. Steinetz, et al., 1960, Endocrinology 67:102-115;

24. Tam, et al., 1991, J. Am. Chem. Soc. 113:6657-6662;

25. Tashima, et al., 1995, J. Clin. Endocrinal. Metab. 80:707-710; and

26. Yang, et al., 1992, Endocrinology 130:179-185.

1. A method of decreasing collagen synthesis, comprising: administeringa synthetic relaxin-like factor to cells of a human, wherein said cellsexpress relaxin receptors; and allowing the synthetic relaxin-likefactor to contact the receptors for a period of time and underconditions such that the receptors are activated, and collagen synthesisis decreased; the synthetic relaxin-like factor comprising an A chainand a B chain, said A chain having the amino acid sequence:Ala-Ala-Ala-Thr-Asn-Pro-Ala-Arg-Tyr-Cys-Cys-Leu-Ser-Gly-Cys-Thr-Gln-Gln-Asp-Leu-Leu-Thr-Leu-Cys-Pro-Tyr(SEQ ID NO:3) or said amino acid sequence (SEQ ID NO:3) truncated by upto about 6 amino acids from the N terminus and/or by up to 6 amino acidsfrom the C-terminus; said B chain having the amino acid sequence:Pro-Thr-Pro-Glu-Met-Arg-Glu-Lys-Leu-Cys-Gly-His-His-Phe-Val-Arg-Ala-Leu-Val-Arg-Val-Cys-Gly-Gly-Pro-Arg-Trp-Ser-Thr-Glu-Ala(SEQ ID NO:4) or said amino acid sequence (SEQ ID NO:4) truncated by upto 5 amino acids from the N-terminus and/or by up to 5 amino acids fromthe C-terminus; said A and B chains linked by disulfide bonds betweenamino acid residue number 11 of SEQ ID NO:3 and amino acid number 10 ofSEQ ID NO:4.
 2. The method of claim 1, wherein the syntheticrelaxin-like factor is attached to a detectable label.
 3. The method ofclaim 1, wherein the synthetic relaxin-like factor is chemicallysynthesized.
 4. The method of claim 1, wherein the syntheticrelaxin-like factor is recombinantly produced.